if1 connects obesity and insulin resistance through ......2020/09/24 · on hfd) by peritoneal...

IF1 connects obesity and insulin resistance through mitochondrial reprogramming in association with ANT2

Ying Wang1#, Yaya Guan1#, Jiaojiao Zhang1, Xinyu Cao2, Shuang Shen2, Genshen Zhong1, Xiwen Xong3, Yanhong Xu2, Xiaoying Zhang2, Hui Wang1* and Jianping Ye2,4*

1 Henan Key Laboratory of Immunology and Targeted Therapy, Henan Collaborative Innovation Center of Molecular Diagnosis and Laboratory Medicine, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang 453003, Henan, China. 2 Shanghai Diabetes Institute, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, Shanghai 201306, China.

3 School of Forensic Medicine, Xinxiang Medical University，Xinxiang 453003, China. 4 Central laboratory, Shanghai Sixth People’s Hospital East Campus, Shanghai Jiao Tong University, Shanghai 201306, China. *Correspondence: Jianping Ye: [email protected]; Hui Wang: [email protected] # Contribute equally to this study. Word counts:

Abstract: 278

Full text: 4578

Reference: 41

Running title: IF1 in obesity

Keywords: Obesity, insulin resistance, ATP synthase inhibitor 1, mitophagy, ANT2

Financial support: This study was funded by the National Key R&D Program of China (2018YFA0800603), and the financial support of the Shanghai Jiao Tong University Affiliated Sixth People’s Hospital and Xinxang Medical University to JY.

.CC-BY 4.0 International licenseavailable under a(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprintthis version posted September 25, 2020. ; https://doi.org/10.1101/2020.09.24.311076doi: bioRxiv preprint

https://doi.org/10.1101/2020.09.24.311076http://creativecommons.org/licenses/by/4.0/

Abstract

IF1 (ATPIF1) is a nuclear DNA-encoded protein with an activity in the inhibition of catalytic

activity of F1Fo-ATP synthase (ATPase), an enzyme for ATP synthesis in mitochondria. A role

of IF1 remains unknown in the metabolic disorder in obesity. In this study, IF1 was examined in

the diet-induced obese (DIO) mice and a decrease in IF1 protein was observed in several tissues

including the skeletal muscle, liver and intestine in the absence of mRNA alteration. Significance

of the reduction was investigated in the IF1-KO mice, in which insulin sensitivity was improved

in the absence of body weight alteration on Chow diet. On a high fat diet (HFD), the IF1-KO

mice gain more body weight as a result of enhanced fat tissue growth. The energy expenditure

and locomotion activity were decreased in the KO mice without an alteration in food intake. The

increase in insulin sensitivity remained in the obese KO mice. The colon tissue exhibited a

resistance to the HFD-induced atrophy with less cell apoptosis and more secretion of GLP-1.

Mitochondria exhibited an enhanced ATP production and maximal oxygen consumption without

an alteration in the respiratory chain proteins. However, the ATP level was reduced in the fasting

condition in the muscle as well as the liver. Mitophagy was enhanced with elevated

accumulation of PINK1 and Parkin proteins in the mitochondria. The protein abundance of

ADP/ATP translocase 2 (ANT2) was decreased in the inner membrane of mitochondria to

account for the reduced apoptosis and enhanced mitophagy. The data suggest that the IF1

reduction in obesity leads to reprogramming of mitochondrial metabolism in a compensatory

response to maintain the insulin sensitivity through down-regulation of ANT2 protein.




INTRODUCTION

Obesity increases the risk of type 2 diabetes through induction of insulin resistance. Energy

surplus leads to insulin resistance through suppression of the insulin signaling activity in cells

under obesity. The signal of energy surplus remains unknown for the suppression although

several candidate molecules have been widely reported, such as ATP, reactive oxygen species

(ROS), diglyceride, ceramide, glucose, and free fatty acids, etc. [1; 2]. However, translation of

those findings into therapy of type 2 diabetes remains a challenge in the drug discovery field.

Inhibition of ATP production represents a mechanism for the therapeutic activity of metformin in

the type 2 diabetes patients, which is associated with activation of AMPK pathway in cells [3].

Inhibition of ATP production with chemical uncouplers [4; 5] or berberine [6; 7] is another

example for the role of ATP in the mechanism of insulin resistance. These lines of evidence

suggest that a surplus in ATP supply is a potential risk factor in the pathogenesis of insulin

resistance. However, the possibility remains to be tested in vivo, especially in a transgenic model.

The intracellular ATP level is determined by the balance of production and consumption in cells.

The ATP level is maintained at a “set point” level, which varies in cell types for around 5-7 mM

in the cardiomyocytes [8]. An increase in ATP consumption leads to more production by the

mitochondria to maintain the cytoplasmic ATP hemostasis [9]. Mitochondria activity in ATP

production is dependent on the oxidative phosphorylation reaction, which is conducted by the

five complexes in the respiratory chain. The F1Fo-ATP synthase (ATPase) in the complex V has

a well-known activity in the production of ATP through phosphorylation of ADP under

conditions of sufficient supply of substrates including oxygen, glucose, fatty acids and amino

acids. However, the ATPase also hydrolyzes ATP in the conditions of substrate deficiency in an




effort to maintain the mitochondrial membrane potential [10]. The hydrolytic activity of ATPase

may prevent ATP surplus in cells in favor of insulin sensitivity. The ATPase activities are

controlled by the inhibitor protein IF1, which inhibits both activities of ATPase [10]. In the

absence of IF1, the activities of ATPase are enhanced in the production as well as hydrolysis of

ATP. An impact of such reprogramming of ATPase activities in insulin sensitivity remains to be

tested after IF1 inhibition, especially following the enhanced hydrolytic activity of ATPase. To

address this issue, we conducted this study in IF1-KO mice, which included both male and

female mice.

We examined the IF1 in the DIO mice and observed reduction in the protein in the muscle, liver

and intestine. A role of the reduction was examined in the IF1-KO mice, in which obesity was

enhanced without an extra impairment in insulin sensitivity. The phenotype was associated with

mitochondrial reprogramming in energy metabolism and quality control, which was observed

with a reduction in ANT2 protein.

RESEARCH DESIGN AND METHODS

Generation of IF1-KO mice and DIO mice

All of the animal procedures were approved by the Institutional Animal Care and Use Committee

(IACUC) of the Shanghai Jiao Tong University. The IF1 (ATPIF1) knockout mouse was

generated in C57BL/6 mice with the CRISPR/Cas9 gene editing method with sgRNA PAM

sequences targeting ATPIF1: SgRNA-1 GCAGTCGGATAGCATGGATACGG and SgRNA-2

GGCTCCACCAGCTTCTCGGATGG. For identification of IF1-/- mice, PCR was conducted to

amplify the IF1 gene using the forward primer 5-CATCAGCCTTGGAATTCTGC-3 and the




reverse primer 5-CTTCGTCTCGGACTCGGTAG-3. The agarose electrophoresis was

performed to determine the genotype, and the amplified PCR product was used in gene

sequencing with ABI-3730XL to confirm the genotype. The IF1 protein was examined to

confirm the gene knockout. The mouse housing environment includes a 12:12-hr light-dark cycle,

constant room temperature (22–24°C), free access to water and diet. The mice were fed on Chow

diet (Fat content ≥ 4% in weight, P1101F-25, Shanghai Pluteng Biotechnology Co., Ltd., China)

or the high fat diet (HFD, Fat = 40% in kcal, D12108C, Jiangsu Synergy, China). HFD feeding

was started at 8 wks in age to generate the diet-induced obese (DIO) model. Age- and gender-

matched wild type mice were used in the control for the IF1-KO mice.

Body weight, body composition and food intake

Body weight of mice was measured weekly. The body composition was determined using the

quantitative nuclear magnetic resonance (NMR) (Minispec Mn10 NMR scanner,

Brucker, Milton, ON, Canada) in the conscious and unrestrained mice, which was placed

individually in a small tube for the NMR analyzer with 1 min in the assay. Food intake was

measured manually or with the metabolic chamber individually on HFD. A mean value of daily

food intake was determined over 3 days. Unit of food intake was g/mouse/day.

Energy expenditure and physical activity

Energy metabolism was monitored in the mice after 4 wks on HFD, using the 20-cage

Promethion-C continuous, parallel metabolic phenotyping system (Sable Systems International,

Las Vegas, USA). Mice were kept in the metabolic chamber individually for 6 days. The oxygen

consumption (VO2), carbon dioxide production (VCO2), spontaneous physical activity and food

intake were recorded daily for 5 days. The data on day 5 were used in the calculation of energy

expenditure (EE: kcal/kg/h) and physical activity. The calculation was performed with the




formula EE = [3.815 + 1.232 × VCO2/VO2] × VO2 × 0.001 [11]. Energy expenditure data was

normalized with the body lean mass.

ITT and GTT

Intraperitoneal insulin tolerance test (ITT) was performed in mice (9 wk on Chow diet and 14 wk

on HFD) by peritoneal insulin injection (0.75 U/kg body weight, I9278, Sigma) after 8 hr fasting.

In DIO mice, oral glucose tolerance test (oGTT) and intraperitoneal glucose tolerance test

(ipGTT) were performed in the mice (12-16 wks on HFD) by administration of glucose (2 g/kg

body weight) after overnight fasting. Blood glucose was measured in the tail vein blood at 0, 15,

30, 60, 90 and 120�min using the Accu-CHEK Advantage blood glucose meter (Accu-CHEK;

Indianapolis, IN). Data were expressed in blood glucose concentration (mmol/l, mM).

Western blotting

Tissues (liver, muscle and colon) were collected from the mice after 16 wks on HFD and

examined for IF1 protein, insulin signaling, apoptosis and mitophagy. The whole cell lysates

were prepared from the tissue in Western blotting according to the protocols described elsewhere

[11]. Antibodies to ATPIF1 (ab110277), VDAC1 (ab14734), PINK1 (ab186303), Parkin

(ab77924), total OXPHOS Rodent (ab110413), P62 (ab56416), β-actin (ab8224) and GAPDH

(ab7291) were obtained from Abcam (Cambridge, MA, USA). Antibodies to Agt7 (8558s),

ANT2 (14671S), Caspase 3 (9662S), cleaved caspase 3 (9664S), Akt (C67E7) and p-Akt (T308,

C31E5E) were purchased from the Cell Signaling Technology (Boston, USA). GAPDH and β-

actin were used as the internal controls. Western blot images were quantified with the ImageJ

software, whose value is expressed by integrated optical density (IOD).

Quantitative real time PCR (qRT-PCR)




The total mRNA was extracted from the tissue using TRIzol reagent according to the

manufacturer’s protocol (TAKARA, Japan). The qRT-PCR assay was performed with a kit of

TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) (TAKARA, Japan) using the ABI-3730XL

machine. The target mRNA was normalized to the ribosome 18S RNA of the endogenous control.

Primers and probes were purchased from TaKaRa (RR820A, Nojihigashi, Kusatsu, Shiga, Japan).

Sequence of GLP-1 (Gcg) green primers include forward primer 5-

TTACTTTGTGGCTGGATTGCTT-3, and reverse primer 5-AGTGGCGTTTGTCTTCATTCA-

3. The primers of IF1 are forward: 5-ACGGGCGCTGGCTCCATCC-3; reverse: 5-

TGGCGTTCAATTTGCTTCTG-3.

Insulin and GLP-1 assay

Insulin and GLP-1 were tested in the plasma of ocular vein blood in mice at 16 wks on HFD.

Plasma insulin was measured using an insulin ELISA Kit (90080, Crystal Chem, Downers Grove,

USA). The blood for GLP-1 assay was collected with a heparin tubes containing dipeptidyl

peptidase IV inhibitor (10 μl/mL), which was conducted at 15 min after the glucose

administration. The active form of GLP-1 was measured with the GLP-1 ELISA Kit (E-EL-

M0090c, Elabscience, Wuhan, China).

HE staining and transmission electron microscopy

The fresh intestinal tissues were fixed in 4% paraformaldehyde for 24 h in the HE staining, and

the electron microscope fixative at 4 � for 2 h in the electron microscopy study. The samples

were prepared by the Wuhan Servicebio technology Co., LTD.

Mitochondrial and ATP assays

The mitochondria were isolated from the fresh muscle tissue of mouse using a protocol modified

from our early study and used in the functional assay with the Agilent Seahorse XF24 Analyzer




equipment (Agilent，Santa Clara, California, USA) [12]. When the muscle tissue was used in

the mitochondrial assay, the fresh tissue was washed with the Agilent Seahorse XF-DMEM

medium (the assay medium) containing 2 mM L-glutamine, 1 mM sodium pyruvate and 10 mM

glucose to remove the non-fatty substances. A piece of tissue (2 × 2 × 1 mm) was placed in each

well of the Agilent Seahorse XF24 islet capture microplate (103518-100，Agilent，Santa Clara,

California, USA) and covered with an islet capture screen to allow free perfusion while

minimizing tissue movement. The oxygen consumption rate (OCR) was determined in the

Agilent Seahorse XF Complete Assay Medium (500 μL) with the Agilent Seahorse XF24

Analyzer. Following agents were used in the stimulation or inhibition of mitochondrial activity

in the assay: ADP (4 mM, A5285, Sigma), Oligomycin (Oligo, 1 μM, 495455, Sigma), FCCP (2

μM, C2920, Sigma), and Antimycin A (AA, 0.5 μM, 2247-10, Biovision). The tissue ATP

concentration was determined in the homogenization of fresh tissues using the EnzyLightTM

ATP Assay Kit (EATP-100, BioAssay Systems, Northern California, USA).

Statistical Analysis. Statistical analysis was performed using two-tailed, unpaired Student’s t-

test in the study. p < 0.05 was considered significant. Results are presented as mean ± SD.

RESULTS

IF1 protein is decreased in multiple tissues of DIO mice

IF1 is a mitochondrial protein that associates with F1Fo-ATPase to inhibit the catalytic activities.

There was no systematic study of distribution of IF1 protein in the mouse tissues when this

project was initiated. We addressed this issue by examining IF1 protein in 9 major tissues of

mice with normalization to the internal control of GAPDH protein. The protein levels from high




to low were in the following order: brown fat, heart, kidney, liver, spleen, lung, colon, brain, and

skeletal muscle (Fig. 1A). An impact of obesity in the IF1 protein was investigated in DIO mice

to understand the relationship of IF1 and obesity. The IF1 protein was examined in the insulin

sensitive tissues including brown and white fat, skeletal muscle and liver tissues. A dramatic

decrease was observed in the IF1 protein in all tissues (Fig. 1, B-D). In addition, the IF1 protein

was reduced by obesity in the colon (Fig. 1E). mRNA of IF1 was not altered in the DIO mice

(Fig. 1E). The results suggest that IF1 protein was expressed in all tissues and the protein

abundance was reduced by obesity.

ATP production and mitochondrial spare capacity are enhanced in mitochondria of IF1-

KO mice

To understand the biological significance of IF1 reduction, we generated the IF1-KO mice with

successful deletion of IF1 protein in the mice (Fig. 2A). The mitochondrial oxygen consumption

rate (OCR) was examined in the skeletal muscle of KO mice to evaluate the impact of IF1

inactivation in mitochondrial function. The study was conducted in isolated mitochondria in

response to challenges of ADP and uncoupler FCCP using the Seahorse equipment. The

mitochondria exhibited an increase in the respiration as indicated by the elevated OCR at the

basal and challenged conditions (Fig. 2, B and C). The protein abundance was not significantly

altered in the respiratory chain of mitochondria in the IF1-KO mice (Fig. 2D). The data suggest

that in the absence of IF1, the production of ATP upon ADP challenge is enhanced for the

elevated activity of ATPase. The alteration leads to an increase in the mitochondrial spare

capacity as indicated by the elevation in the maximal OCR upon FCCP challenge.




Insulin sensitivity is enhanced in IF1-KO mice on Chow diet

The IF1 protein reduction was associated with insulin resistance in the DIO mice. However, the

cause/effect relationship was unknown for the two events. To address this issue, insulin

sensitivity was examined in the IF1-KO mice on Chow diet in both genders. The insulin

tolerance test was conducted with intraperitoneal injection of insulin (ipITT). The IF1-KO mice

exhibited a significant increase in insulin sensitivity as indicated by more reduction in the blood

glucose in the KO mice over the WT mice (Fig. 3, A and B). There was no significant difference

in the body weight between the KO and WT mice (Fig. 3, C and D). The data suggest that

inhibition of IF1 activity by gene knockout led to an increase in insulin sensitivity in the IF1-KO

mice.

IF1-KO mice gains more fat tissue on HFD

Obesity was induced in the IF1-KO mice with HFD feeding to test the mice responses to the

energy surplus. The IF1-KO mice gained more body weight than the WT mice in both gender

(Fig. 4, A and B). The initial body weight was identical in the female mice, but modestly higher

in the male mice before the feeding. The KO mice exhibited a higher gain in the body weight

than WT mice throughout the study. A difference in the fat accumulation contributed to the extra

weight gain in the KO mice as the total body fat mass, epididymal fat pad, and liver size were all

higher in the KO mice (Fig. 4, C and D). Energy expenditure were examined using the rodent

metabolic chamber. A reduction was observed in the IF1-KO mice at the day- and night-time

(Fig. 4E). A reduction in the physical activity was observed, but a significance was only obtained

at the nighttime (Fig. 4F). Food intake was not significantly altered in the mice (data not shown).




These data suggest that inactivation of IF1 gene led to a reduction in energy expenditure for an

increased risk of obesity in the IF1-KO mice.

Heat production is increased in the IF1-KO mice

The reduction in energy expenditure was observed in the female KO mice in above experiment.

The same experiment was conducted in the male mice. A similar reduction was observed in the

energy expenditure and physical activity in the male mice (Fig. 5, A-D). The reduction was

observed at the nighttime, which corresponded to the reduced oxygen consumption rate at night.

However, food intake was not decreased in the presence of lower physical activity (Data not

shown). Interestingly, heat production was significantly increased in the male IF1-KO mice (Fig.

5, E and F). These results suggest that the reduction in physical activities contributes to the low

energy expenditure in the KO mice. In the energy expenditure, more heat was produced in the

KO mice.

Insulin resistance is disassociated with obesity in the IF1-KO mice

In the IF1-KO mice, insulin sensitivity was examined to determine the impact of obesity in the

glucose metabolism. The study was conducted in several tests including the intraperitoneal

glucose tolerance (ipGTT), insulin tolerance (ipITT) and insulin signaling tests. The KO mice

exhibited an improved insulin sensitivity in all the assays (Fig. 6, A-D). In the GTT assay, the

blood glucose was increased much less in the KO mice (Fig. 6A). In the ITT assay, the blood

glucose was decreased more in the KO mice (Fig. 6B). The insulin signaling pathway was

examined in the skeletal muscle and liver tissues of KO mice with Akt phosphorylation. An

increase was observed in both tissues (Fig. 6, C and D). The ATP level was decreased in the




muscle and liver of IF1-KO mice in the fasting condition (Fig. 6, E and F). This group of data

suggests that insulin sensitivity was not preserved in the KO mice in the presence of increased

adiposity. The phenotype suggests a disassociation of insulin resistance and obesity in the KO

mice.

Plasma GLP-1 is elevated in the IF1-KO mice

Above data suggest that glucose metabolism was reprogrammed in the IF1-KO mice to preserve

insulin sensitivity. The impact of mitochondrial reprogramming in GLP-1 activity was

investigated to understand the contribution of endocrine activity to the glucose homeostasis.

GLP-1 acts in multiple tissues in the regulation of blood glucose in the control of type 2 diabetes.

GLP-1 activity was examined in the KO mice on HFD through an oral glucose tolerance test

(OGTT), which induces GLP-1 secretion in the intestine. OGTT was significantly improved in

the IF1-KO mice (Fig. 7A). Plasma GLP-1 level was examined during the OGTT test, and an

increase was observed in the IF1-KO mice (Fig. 7B). Expression of GLP-1 was examined in

mRNA and a significant elevation was observed in the large intestine of the IF1-KO mice (Fig.

7C). In the DIO mice, GLP-1 secretion is decreased in the large intestine from a tissue atrophy

following mitochondrial degeneration [12]. The colon tissues were examined in the IF1-KO mice

to understand the mechanism of improved GLP-1 secretion. The thickness of intestine wall and

mucosa layer were measured in the evaluation of intestine atrophy after H&E staining. An

increase in the thickness was observed in the IF1-KO mice with abundant smooth muscle and

mucosa content (Fig. 7D). These data suggest that GLP-1 secretion was improved in the intestine

of IF1-KO mice, which may contribute to the disassociation of insulin resistance and obesity in

the obese IF1-KO mice.




Mitochondria exhibits an improved quality control in the IF1-KO mice

Villi and mitochondrial morphology were examined in the colonic epithelial cells under the

electron microscope. The villi were heathier in the IF1-KO mice with persistent length versus the

sickness type of villi of different length in the WT mice (Fig. 8A). In the super structure,

mitochondria had smaller size and higher matrix density in the epithelial cells of the IF1-KO

mice (Fig. 8A), which are signs of healthy mitochondria. In contrast, mitochondria of the WT

mice had a larger size and less crista in the parameters of mitochondrial degeneration.

Mitochondria control tissue atrophy through an impact in apoptosis. The mitochondrial alteration

may reduce tissue atrophy through downregulation of apoptosis in the IF1-KO mice. To test the

possibility, the cleaved caspase 3 (c-Cas3) signal was examined to determine apoptosis, and a

reduction was observed in the large intestine of IF1-KO mice (Fig. 8B). Adenine nucleotide

translocase 2 (ANT2), a ADP/ATP translocase in the mitochondrial inner membrane, contributes

to apoptosis by mediating proton leak in the collapse of mitochondrial potential [13]. A decrease

in ANT2 activity leads to less proton leak in the inhibition of cell apoptosis [14; 15]. ANT2

protein was significantly reduced in the colon tissue of IF1-KO mice (Fig. 8B). Mitophagy

markers, PINK1 and Parkin, were examined to determine the mechanism of mitochondrial

activity in apoptosis. Both signals were increased in the IF1-KO mice colon (Fig. 8C). The

autophagy markers including p62 and Atg7 were not changed in the same condition. Under the

electronic microscope, mitophagy was observed with mitochondrial structure in the

autophagosome (Fig. 8B), in which more mitochondria were found in the autophagosome of IF1-

KO mice as pointed by the white color arrows. These data suggest that the reduction in tissue




atrophy may be a result of suppression of cell apoptosis following the decrease in the ANT2

protein together with the increased mitophagy activity.

DISCUSSION

The study demonstrates that the IF1 protein is expressed in all tissues, and the abundance is

reduced by obesity. There was no information about regulation of IF1 protein in the normal

tissues in the literature except tumor tissue [16; 17], in which IF1 protein is increased to promote

glycolysis in certain types of tumor [16; 18]. In this study, the IF1 protein was identified in all

tissues with the highest level in the heart muscle, kidney and brown fat of mice, which is

consistent with the mRNA expression level in those tissues in a recent study [19]. There is very

limited information about regulation of IF1 protein by posttranslational modification. IF1 is

subject to phosphorylation by PKA in the functional inhibition and modification by proton for

activation in the acidic condition of mitochondria [19; 20]. However, an impact of those

modifications in the protein stability remains unknown [17]. Mitochondrial proteins are studied

in the human skeletal muscle of obese patients with the proteomics technology [21]. However,

the study does not identify a change in the IF1 protein. The IF1 protein was examined by

Western blotting in a study of the skeletal muscle, and an increase was observed in the obese

subjects [22], which is opposite to ours. The cause for the difference is unknown. The difference

in the model systems may play a role. As mRNA of IF1 was not changed, our data suggests that

the reduction in IF-1 protein is a result of translational regulation or post-translational

modification, which is supported by the feature of mitochondrial proteins [21; 23]. The exact

mechanism remains to be investigated for the IF1 protein reduction.




Our study reveals that a metabolic reprogramming is induced in mitochondria by IF1 inactivation.

The biological significance of the IF1 reduction was investigated in mitochondria of skeletal

muscle, liver and intestine. The ATP production and the maximal oxygen consumption were all

elevated in the isolated mitochondria of the IF1-KO mice. The increased ATP production reflects

an elevation in the synthetic activity of ATPase enzyme in the absence of IF1 [10], which

associates with the increase in the maximal oxygen consumption in response to the uncoupler

FCCP. In the fasting condition, ATP level was decreased in the skeletal muscle and liver of IF1-

KO mice, which is consistent with the increased hydrolytic activity of ATPase in the absence of

IF1 [10]. The metabolic reprogramming was not associated with a change in the protein

abundance of the respiratory chain. These data suggest that the elevation in catalytic activities of

ATPase is a key in the metabolic reprogramming in mitochondria.

The study demonstrates that the mitochondrial reprograming increased the risk of obesity in the

IF1-KO mice, which was observed in both male and female mice. There are two potential

mechanisms for this obesity phenotype. The first is reduction in the locomotion activity in the

IF1-KO mice for the less energy expenditure as indicated by the fall in oxygen consumption,

which was observed in the mice on HFD in the metabolic chamber. The reduction did not lead to

a decrease in the food intake in the IF1-KO mice. The second mechanism is an enhancement of

adipocyte differentiation that was observed in vitro for an increased accumulation of triglyceride

and enlarged lipid droplets in differentiated adipocytes from preadipocytes of the IF1-KO mice

[24]. The obesity is supported by the phenotypes of IF1 overexpression mice, in which ROS

production is enhanced [25] and inflammatory response is elevated [26]. ROS and inflammation

promote energy expenditure in favor of suppression of obesity [27].




The mitochondrial reprograming disassociated obesity and insulin resistance in the IF1-KO mice

of both genders. In the WT mice, obesity is positively associated with insulin resistance.

However, this rule was broken in the IF1-KO mice on HFD, which exhibited more adiposity

without extra impairment in insulin sensitivity. On the Chow diet, the IF1-KO mice exhibited no

difference in the body weight, but a better insulin sensitivity over the WT mice. The IF1-KO

mice were normal in the growth, development and reproduction on the Chow diet, which is

consistent with that in a study by Nakamura [28]. Obesity and insulin sensitivity were not

examined in the IF1-KO mice in Nakamura’s study. Our data suggest that IF1 may inhibit insulin

sensitivity in the physiological condition, which was abolished in the IF1-KO mice.

The mitochondrial reprograming increased GLP-1 secretion, which provides an endocrine

mechanism for the enhanced glucose metabolism in the IF1-KO mice. There is no report about

the relationship of IF1 and GLP-1 in the literature. In the WT mice, GLP-1 secretion is reduced

by HFD through a tissue atrophy in the intestine [12], which is associated with mitochondrial

degeneration and cell apoptosis [29]. In the IF1-KO mice, both pathological alterations were

prevented by the mitochondrial reprogramming. Consistently, IF1 inhibition by gene knockdown

led to inhibition of apoptosis through stabilization of mitochondrial potential [30]. However, the

mitochondrial membrane permeability was not investigated in that study. We addressed this

issue by testing ANT2 in the IF1-KO mice.

The mitochondrial reprograming was associated with ANT2 reduction in the IF1-KO mice.

ANT2 is a transporter protein located in the inner membrane of mitochondria to mediate the




import of ADP from the cytoplasm and export of ATP from mitochondria [31]. ANT2 belongs to

the ANT family whose activity is regulated by the Bcl family proteins in the control of apoptosis

[13]. However, the exact action remains unknown for the ANT proteins until that ANT1 was

found to transport proton in a study of mitochondrial uncoupling activity [32]. An increase in the

ANT activity makes the cells more susceptible to apoptosis for a risk of the mitochondrial

potential collapse [14]. It was unknown how IF1 regulates ANT in mitochondria. In current

study, the ANT2 activity was reduced in the IF1-KO mice, which provides a mechanism for the

resistance to apoptosis in support of the GLP-1 secretion function.

The ANT2 reduction may contribute to the enhanced insulin action through mitophagy in the

IF1-KO mice. ANT2 gene knockout improves insulin sensitivity in two studies of fat and liver in

mice [33; 34]. The exact mechanism remains to be investigated for the enhanced insulin action

although a reduction in inflammation was proposed [33]. Mitophagy removes the senescent and

damaged mitochondria through self-digestion [35], a process triggered by multiple factors

including energy deficiency [36]. In current study, mitophagy was enhanced in the IF1-KO mice,

which is consistent with the phenotypes after IF1 inhibition by shRNA-mediated gene

knockdown [25] or gene knockout [37]. However, the role of IF1 in mitophagy remains to

established as IF1 knockdown inhibited mitophagy in a cellular model for a deficiency in Parkin

(Park2) recruitment [38]. The increase in mitophagy and insulin sensitivity provides a

mechanism for the enhanced lipid accumulation in white adipocytes underlying the increased

obesity in IF1-KO mice in our study. Mitophagy reduces the mitochondrial number in adipocytes

to decrease energy expenditure in support of triglyceride storage [39]. Insulin stimulates

triglyceride synthesis and storage in adipocytes. These activities may attenuate the GLP-1 effect




in the control of body weight in the IF1-KO mice. The enhanced mitophagy provides a unified

mechanism for the enhanced obesity, insulin sensitization and resistance to apoptosis in the IF1-

KO mice. Inhibition of autophagy has opposite effects on metabolism as documented in reviews

[35; 40]. The IF1 activity is likely mediated by the reduction in ANT2 activity. An enhanced

mitophagy is reported in the presence of ANT inhibition by a pharmacological agent [41]. ANT2

reduction may contribute to the control of ATP homeostasis by reducing ADP supply to

mitochondria. The mechanism of ANT2 reduction in the IF1-KO mice remains to be investigated.

In summary, the role of IF1 protein is investigated in the pathogenesis of obesity in this study.

IF1 protein was reduced in mitochondria by obesity, which represents a compensatory response

to the energy surplus in obesity. The conclusion is supported by the metabolic phenotype of IF1-

KO mice in both male and female mice, in which obesity was enhanced with a disassociation to

insulin resistance. The mitochondrial reprogramming is an underlying mechanism for the

enhanced insulin sensitivity and enhanced GLP-1 secretion. The reprogramming was observed in

the mitochondrial function and structure in IF1-KO mice. The ATPase activity was enhanced,

but ANT2 activity was reduced to limit the ADP supply in mitochondria in the maintenance of

ATP homeostasis. The reprogramming was observed with an increase in mitophagy. ANT2 may

mediate the IF1 activity in the mitochondrial reprogramming. The molecular mechanism of

ANT2 reduction remains to be investigated in the IF1-KO mice.

Author contribution: Y.W, Y.G, J.Z, X.C, S.S, G.Z, X.X, and X.Z. conducted the experiments.

H.W and J.Y designed the study and prepared the manuscript. All authors read and approved the

final manuscript.




Acknowledgment: This study was funded by the National Key Research and Development

Program of China (2018YFA0800603 to JY). The authors have no conflict of interest in the

publication of this study.




Reference

[1] Ye, J., 2013. Mechanisms of insulin resistance in obesity. Front Med 7(1):14-24. [2] Roden, M., Shulman, G.I., 2019. The integrative biology of type 2 diabetes. Nature

576(7785):51-60. [3] Rena, G., Hardie, D.G., Pearson, E.R., 2017. The mechanisms of action of metformin.

Diabetologia 60(9):1577-1585. [4] Perry, R.J., Kim, T., Zhang, X.M., Lee, H.Y., Pesta, D., Popov, V.B., et al., 2013.

Reversal of hypertriglyceridemia, fatty liver disease, and insulin resistance by a liver-targeted mitochondrial uncoupler. Cell Metab 18(5):740-748.

[5] Jiang, H., Jin, J., Duan, Y., Xie, Z., Li, Y., Gao, A., et al., 2019. Mitochondrial Uncoupling Coordinated With PDH Activation Safely Ameliorates Hyperglycemia via Promoting Glucose Oxidation. Diabetes 68(12):2197-2209.

[6] Yin, J., Gao, Z., Liu, D., Liu, Z., Ye, J., 2008. Berberine Improves Glucose Metabolism through Induction of Glycolysis. Am J Physiol Endocrinol Metab 294:E148-E156.

[7] Turner, N., Li, J.Y., Gosby, A., To, S.W., Cheng, Z., Miyoshi, H., et al., 2008. Berberine and its more biologically available derivative, dihydroberberine, inhibit mitochondrial respiratory complex I: a mechanism for the action of berberine to activate AMP-activated protein kinase and improve insulin action. Diabetes 57(5):1414-1418.

[8] Allue, I., Gandelman, O., Dementieva, E., Ugarova, N., Cobbold, P., 1996. Evidence for rapid consumption of millimolar concentrations of cytoplasmic ATP during rigor-contracture of metabolically compromised single cardiomyocytes. Biochem J 319 ( Pt 2):463-469.

[9] Wilson, D.F., 2017. Oxidative phosphorylation: regulation and role in cellular and tissue metabolism. J Physiol 595(23):7023-7038.

[10] Cao, X., Zhang, X., Ye, J., 2020. Regulation of Mitochondrial ATP Synthase Inhibitor 1 in Cellular Energy Metabolism. Chinese Journal of Cell Biology 42(4):682-690.

[11] Ke, B., Zhao, Z., Ye, X., Gao, Z., Manganiello, V., Wu, B., et al., 2015. Inactivation of NF-kappaB p65 (RelA) in liver improved insulin sensitivity and inhibited cAMP/PKA pathway. Diabetes 64(10):E496-505.

[12] Le, J., Zhang, X., Jia, W., Zhang, Y., Luo, J., Sun, Y., et al., 2019. Regulation of microbiota-GLP1 axis by sennoside A in diet-induced obese mice. Acta Pharm Sin B 9(4):758-768.

[13] Zhivotovsky, B., Galluzzi, L., Kepp, O., Kroemer, G., 2009. Adenine nucleotide translocase: a component of the phylogenetically conserved cell death machinery. Cell Death Differ 16(11):1419-1425.

[14] Qin, X., Xu, Y., Peng, S., Qian, S., Zhang, X., Shen, S., et al., 2020. Sodium butyrate opens mitochondrial permeability transition pore (MPTP) to induce a proton leak in induction of cell apoptosis. Biochem Biophys Res Commun 527(3):611-617.

[15] Wu, K., Luan, G., Xu, Y., Shen, S., Qian, S., Zhu, Z., et al., 2020. Cigarette smoke extract increases mitochondrial membrane permeability through activation of adenine nucleotide translocator (ANT) in lung epithelial cells. Biochem Biophys Res Commun 525(3):733-739.

[16] Sanchez-Arago, M., Formentini, L., Martinez-Reyes, I., Garcia-Bermudez, J., Santacatterina, F., Sanchez-Cenizo, L., et al., 2013. Expression, regulation and clinical relevance of the ATPase inhibitory factor 1 in human cancers. Oncogenesis 2:e46.




[17] García-Aguilar, A., Cuezva, J.M., 2018. A Review of the Inhibition of the Mitochondrial ATP Synthase by IF1 in vivo: Reprogramming Energy Metabolism and Inducing Mitohormesis. Frontiers in Physiology 9:1322.

[18] Sanchez-Cenizo, L., Formentini, L., Aldea, M., Ortega, A.D., Garcia-Huerta, P., Sanchez-Arago, M., et al., 2010. Up-regulation of the ATPase inhibitory factor 1 (IF1) of the mitochondrial H+-ATP synthase in human tumors mediates the metabolic shift of cancer cells to a Warburg phenotype. The Journal of biological chemistry 285(33):25308-25313.

[19] Esparza-Molto, P.B., Nuevo-Tapioles, C., Chamorro, M., Najera, L., Torresano, L., Santacatterina, F., et al., 2019. Tissue-specific expression and post-transcriptional regulation of the ATPase inhibitory factor 1 (IF1) in human and mouse tissues. Faseb J 33(2):1836-1851.

[20] Garcia-Bermudez, J., Sanchez-Arago, M., Soldevilla, B., Del Arco, A., Nuevo-Tapioles, C., Cuezva, J.M., 2015. PKA Phosphorylates the ATPase Inhibitory Factor 1 and Inactivates Its Capacity to Bind and Inhibit the Mitochondrial H(+)-ATP Synthase. Cell Rep 12(12):2143-2155.

[21] Kras, K.A., Langlais, P.R., Hoffman, N., Roust, L.R., Benjamin, T.R., De Filippis, E.A., et al., 2018. Obesity modifies the stoichiometry of mitochondrial proteins in a way that is distinct to the subcellular localization of the mitochondria in skeletal muscle. Metabolism 89:18-26.

[22] Formentini, L., Ryan, A.J., Galvez-Santisteban, M., Carter, L., Taub, P., Lapek, J.D., Jr., et al., 2017. Mitochondrial H(+)-ATP synthase in human skeletal muscle: contribution to dyslipidaemia and insulin resistance. Diabetologia 60(10):2052-2065.

[23] Ruegsegger, G.N., Creo, A.L., Cortes, T.M., Dasari, S., Nair, K.S., 2018. Altered mitochondrial function in insulin-deficient and insulin-resistant states. The Journal of Clinical Investigation 128(9):3671-3681.

[24] Guan, Y., Liang, Y., Wang, H., Ye, J., 2018. Knockout of ATP synthase inhibitory factor 1 (ATPIF1) gene increased intracellular ATP level in primary fibroblast cells and promoted adipocyte differentiation Journal of Xinxiang Medical College 35:658-661.

[25] Campanella, M., Seraphim, A., Abeti, R., Casswell, E., Echave, P., Duchen, M.R., 2009. IF1, the endogenous regulator of the F(1)F(o)-ATPsynthase, defines mitochondrial volume fraction in HeLa cells by regulating autophagy. Biochim Biophys Acta 1787(5):393-401.

[26] Formentini, L., Sánchez-Aragó, M., Sánchez-Cenizo, L., Cuezva, José M., 2012. The Mitochondrial ATPase Inhibitory Factor 1 Triggers a ROS-Mediated Retrograde Prosurvival and Proliferative Response. Molecular Cell 45(6):731-742.

[27] Wang, H., Ye, J., 2015. Regulation of energy balance by inflammation: common theme in physiology and pathology. Rev Endocr Metab Disord 16(1):47-54.

[28] Nakamura, J., Fujikawa, M., Yoshida, M., 2013. IF1, a natural inhibitor of mitochondrial ATP synthase, is not essential for the normal growth and breeding of mice. Biosci Rep 33(5):735-741.

[29] Sun, Y., Jin, C., Zhang, X., Jia, W., Le, J., Ye, J., 2018. Restoration of GLP-1 secretion by Berberine is associated with protection of colon enterocytes from mitochondrial overheating in diet-induced obese mice. Nutrition & Diabetes 8:53.




[30] Faccenda, D., Nakamura, J., Gorini, G., Dhoot, G.K., Piacentini, M., Yoshida, M., et al., 2017. Control of Mitochondrial Remodeling by the ATPase Inhibitory Factor 1 Unveils a Pro-survival Relay via OPA1. Cell Rep 18(8):1869-1883.

[31] Ruprecht, J.J., King, M.S., Zogg, T., Aleksandrova, A.A., Pardon, E., Crichton, P.G., et al., 2019. The Molecular Mechanism of Transport by the Mitochondrial ADP/ATP Carrier. Cell 176(3):435-447 e415.

[32] Bertholet, A.M., Chouchani, E.T., Kazak, L., Angelin, A., Fedorenko, A., Long, J.Z., et al., 2019. H(+) transport is an integral function of the mitochondrial ADP/ATP carrier. Nature 571(7766):515-520.

[33] Seo, J.B., Riopel, M., Cabrales, P., Huh, J.Y., Bandyopadhyay, G.K., Andreyev, A.Y., et al., 2019. Knockdown of Ant2 Reduces Adipocyte Hypoxia And Improves Insulin Resistance in Obesity. Nat Metab 1(1):86-97.

[34] Cho, J., Zhang, Y., Park, S.Y., Joseph, A.M., Han, C., Park, H.J., et al., 2017. Mitochondrial ATP transporter depletion protects mice against liver steatosis and insulin resistance. Nat Commun 8:14477.

[35] Zhang, Y., Sowers, J.R., Ren, J., 2018. Targeting autophagy in obesity: from pathophysiology to management. Nat Rev Endocrinol.

[36] Wang, L., Qi, H., Tang, Y., Shen, H.M., 2019. Post-translational Modifications of Key Machinery in the Control of Mitophagy. Trends Biochem Sci.

[37] Yang, K., Long, Q., Saja, K., Huang, F., Pogwizd, S.M., Zhou, L., et al., 2017. Knockout of the ATPase inhibitory factor 1 protects the heart from pressure overload-induced cardiac hypertrophy. Sci Rep 7(1):10501.

[38] Lefebvre, V., Du, Q., Baird, S., Ng, A.C., Nascimento, M., Campanella, M., et al., 2013. Genome-wide RNAi screen identifies ATPase inhibitory factor 1 (ATPIF1) as essential for PARK2 recruitment and mitophagy. Autophagy 9(11):1770-1779.

[39] Altshuler-Keylin, S., Kajimura, S., 2017. Mitochondrial homeostasis in adipose tissue remodeling. Sci Signal 10(468):eaai9248.

[40] Rovira-Llopis, S., Banuls, C., Diaz-Morales, N., Hernandez-Mijares, A., Rocha, M., Victor, V.M., 2017. Mitochondrial dynamics in type 2 diabetes: Pathophysiological implications. Redox Biol 11:637-645.

[41] Hoshino, A., Wang, W.J., Wada, S., McDermott-Roe, C., Evans, C.S., Gosis, B., et al., 2019. The ADP/ATP translocase drives mitophagy independent of nucleotide exchange. Nature 575(7782):375-379.




LEGENDS

Fig. 1 Reduction of IF1 protein in DIO mice. A. Distribution of IF1 protein in the mouse

tissues including heart, liver, spleen, lung, kidney, brain, muscle, BAT and colon；B. Abundance

of IF1 protein in the skeletal muscle of lean and obese mice；C. Abundance of IF1 protein in the

liver tissue of lean and obese mice；D. Abundance of IF1 protein in the colon tissue of lean and

obese mice；E. mRNA of IF1 in the muscle and liver tissues (n=6). F. Validation of IF1

knockout by protein assay. The assay was conducted in the skeletal muscle (n=4). The data in bar

figure represents mean ± SD. * p

mice; B. ITT in female mice. C. Male mice body weight. D. Female mice body weight. The data

represents mean ± SD (n=6). * p

mice at 30 min after insulin injection (0.75 U/kg body weight) at 16 weeks on HFD (n=3); D.

Insulin signaling in the liver. The test was conducted in the liver under the same condition as the

muscle assay (n=3). E. ATP level in muscle. The tissue ATP level was determined in the skeletal

muscle in the same samples (n=6). F. ATP level in the liver. The ATP level was determined in the

liver tissue in the same samples (n=6). The data represents mean ± SD. * p

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Cont HFD

Re

lative

mR

NA

le

ve

l

IF1 mRNA

00.20.40.60.8

11.21.41.6

WT KO

Re

lative

le

ve

l

***

0

0.2

0.4

0.6

0.8

1

Control HFD

Re

lative

le

ve

l

Muscle

***

Fig .1 IF1 protein expression was decreased in obese mice

IF1

GAPDH

WT KO

F

A

ATPIF1

GAPDH

0

0.5

1

1.5

2

Control HFD

Re

lative

le

ve

l

*

IF1

GAPDH

Control DIO

C

Control DIO

B

0

0.5

1

1.5

2

2.5

3

Heart Liver Spleen Lung Kidney Brian Muscle BAT Colon

Re

lative

le

ve

l

Colo

n

Heart

Liv

er

Muscle

BA

T

Sple

en

Lung

Kid

ney

Bra

in

ATPIF1

GAPDH

0

0.5

1

1.5

2

Control HFD

Re

lative

le

ve

l

***

*

IF1

D

Control DIO

Actin

Muscle IF1IF1 distribution Liver IF1

Colon IF1IF1-KO mice

E

Control DIO




Fig. 2 Inactivation of IF1 enhanced muscle mitochondrial function and insulin sensitivity on Chow diet

-200

-100

0

100

200

300

400

500

600

700

800

2.06 7.55 14.30 21.04 27.79 34.55

OC

R (

pm

ol/m

in/3

μg

)

Time (min)

WT-Muscle KO-Muscle

A B C D

A B

*

*

0

100

200

300

400

500

600

700

800

900

Basal ADP Oligomycin FCCP Antimycin

A

OC

R（

PM

OL/M

IN/3

UG）

WT-Muscle KO-Muscle

CVCⅢCIV

CⅡ

CⅠ

WT KO

C Respiratory chain

0

1

2

3

Re

lative

le

ve

l

WT KO

CV CIII CIV CII CI

VDAC1

*

*




DB CA

0

2

4

6

8

10

12

0 30 60 90 120

Glu

cose（

mM

)

Time（min）

WT KO*

***** ***

****

0

5

10

15

20

25

30

WT KO

We

ight (g

)

WT KO

0

2

4

6

8

10

0 30 60 90 120

Time（min）

WT KO

**

Glu

cose

(m

M)

Female ITTMale ITT Female body weight Male body weight

Fig. 3 Inactivation of IF1 enhanced insulin sensitivity in the KO mice on Chow diet

0

5

10

15

20

25

WT KO

We

igh

t (g

)

WT KO




0

5

10

15

20

25

30

Light Dark

En

erg

y e

xp

en

ditu

re(K

ca

l/kg

/hr)

WT KO

*****

Fig. 4 IF1-KO mice gained more body weight on HFD with less energy expenditure (Wang)

A

E

CB

0

5

10

15

20

25

30

35

40

0 1 2 3 4 5 6 7 8 9 10 11

We

igh

t(g

)

Weeks on HFD

WT KO

**** ** **

** ** ***

*

**

WT KO

gWAT

Liver

Fat

WT KO

D

0

5

10

15

20

25

WT KO

Fat g

ain

per

mo

use

(g) **

F

Body weight of femaleBody weight of male Fat mass of male

0

20

40

60

0 4 8 12 16 20 24

We

igh

t(g

)

weeks on HFD

WT IF1-KO

0 1 2 3 4 5 6 7 8 9 10 11 12

0

200

400

600

800

1000

1200

1400

Light Dark

Am

bula

tory

activity (

me

ter)

WT KO

*




*

Fig. 5 IF1 inactivation led to reduction in ambulatory activity and oxygen consumption with more thermogenesis on HFD

B

C

A

D

E F

11:00 20:00 8:00 11:000

1000

2000

3000

4000

5000WT

KOV

O2(m

l/kg

/h)

11:00 20:00 8:00 11:000

500

1000

1500

2000WT

KO

Am

bu

lato

ry a

cti

vit

y

(be

am

bre

aks)

Dar

k

Ligh

t

Tota

l0

500

1000

1500WT KO

**

Am

bu

lato

ry a

cti

vit

y

(be

am

bre

aks)

11:00 20:00 8:00 11:000.2

0.4

0.6

WT

KO

He

at(

kc

al/

hr)

Dark Light Total0.2

0.4

0.6

0.8

WT KO

** *

He

at(

kc

al/

hr)




A

Akt

pAkt

WT KO

pAkt

WT KO

C

D

Fig. 6 Insulin sensitivity was enhanced in the obese IF1-KO mice (Wang)

B

0

5

10

15

20

25

30

35

0 20 40 60 80 100 120

Glu

cose（

mM

)

Time（min）

WT

KO

*

***

***

**

00.20.40.60.8

11.2

WT KO

pA

kt/

Akt **

0

0.2

0.4

0.6

0.8

1

WT KO

pA

kt/

Akt

*

Akt

Liver

MuscleipGTT

0

0.5

1

1.5

2

2.5

3

WT KO

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

WT KO

AT

P（

µM

/g）

AT

P（

µM

/g）

E F

**

**

Muscle Liver

0

2

4

6

8

10

0 20 40 60 90

Glu

co

se

(m

M)

ITT

Time (min)

KO

WT

*** *




A CB

Fig. 7 Plasma GLP-1 was elevated in the obese IF1-KO mice in OGTT test (Wang)

0

5

10

15

20

25

30

0 20 40 60 80 100 120

Glu

cose（

mM

)

Time（min）

WT

KO

*

****

0

200

400

600

800

1000

1200

1400

WT KO

GLP

-1(n

g m

l-1)

***

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

WT KO

mR

NA

fo

ld c

han

ge

**OGTT Plasma GLP-1 GLP-1 mRNA

IF1-KO

4X 10X 40X

WT

D

500 μm 100 μm

500 μm

50 μm

50 μm100 μm




50

00

X

WT IF1-KO

15

00

0X

30

00

0X

Fig. 8 Tissue and mitochondria in the large intestine of obese IF1-KO mice

A

1 μm 1 μm

500 nm 500 nm

200 nm 200 nm

ANT2

Actin

Cas 3

c-Cas 3

WT KO

B Reduced apoptosis

0

0.2

0.4

0.6

0.8

1

1.2

c-Cas 3 Cas 3 ANT2

Re

lative

le

ve

l WT

KO

* *

D Mitophagosome

WT IF1-KO

GAPDH

Parkin

PINK1

WT KO

P62

Agt7

C Enhanced mitophagy

0

0.5

1

PINK1 Parkin P62 Agt7

Re

lative

le

ve

l

WT KO

**

*

*



if1 connects obesity and insulin resistance through ......2020/09/24 · on hfd) by peritoneal...

Documents